1. Field of the Invention
This invention is in the field of biochemical engineering. More particularly, the invention concerns a pharmaceutical formulation of biologically active recombinant IL-2 or IFN-.beta. protein which is suitable for parenteral injection into humans.
2. Description of Related Disclosures
Naturally occurring interferons (IFNs) are species-specific proteins, often glycoproteins, produced by various cells upon induction with viruses double stranded RNA's, other polynucleotides, antigens and mitogens. Interferons exhibit multiple biological activities such as antiviral, antiproliferative, immunomodulatory and anticellular functions. At least three distinct types of human interferons have been identified and characterized in terms of their anti-viral, anti-growth and activation of natural killer cell (NK) activities. They are produced by leukocytes, lymphocytes, fibroblasts and the immune system and are classified as .alpha., .beta. and .gamma. interferons. These are reported to be different proteins coded for by distinct structural genes.
In recent times, however, several of the human interferon genes have been cloned using recombinant DNA technology and expressed in E. coli (Nagola, S. et al., Nature, 284:316 (1980); Goeddel, D. V. et al., Nature, 287-411 (1980); Yelverton, E. et al., Nuc Acid Res, 9:731 (1981)); and Streuli, M. et al., Proc Natl Acad Sci (US), 78:2848 (1981).
Native human IL-2 is an antigen-nonspecific, genetically unrestricted soluble factor produced by erythrocyte rosette positive T cells stimulated with antigens, mitogens and alloantigens. It is a protein with a reported molecular weight in the approximate range of 13,000 to 17,000 daltons (S. Gillis and J. Watson, J Exp Med (1980) 159:1709) and an isoelectric point in the approximate range of pH 6-8.5. Human IL-2 has a number of in vitro and in vivo effects including enhancing the proliferative responses of human peripheral blood mononuclear cells or murine thymocytes, enhancing the immune response in humans and in animals against bacterial, parasitic, fungal,, protozoan and viral infections, and supporting the growth of continuous T cell lines.
Proteins such as IFNs and IL-2 and muteins thereof in which a cysteine residue is deleted or replaced with another amino acid have been produced microbially through genetic engineering techniques. Microbially produced IL-2 is not glycosylated and is produced in a reduced state by the microorganisms. When purified and oxidized, these microbially produced IL-2s exhibit activity comparable to native human IL-2.
Procedures for purifying native IL-2 from T cells are described by Watson, J., et al, J Exp Med (1979) 150:849-861; Gillis, S., et al, J Immunology (1980) 124:1954-1962; Mochizuki, D. Y., et al, J Immun Meth (1980) 39:185-201; Welte, K., et al, J Exp Med (1982) 156:454-464; and European patent applications 83103582.9 (published Oct. 26, 1983 under no. 92163) and 83400938.3 (published Nov. 16, 1983 under no. 94317). In general, these procedures involve precipitating proteins from culture supernatants with ammonium sulfate followed by a chromatographic fractionation.
Procedures for recovering and purifying bacterially produced IFNs are described in U.S. Pat. Nos. 4,450,103; 4,315,852; 4,343,735; and 4,343,736; and Derynck, R., et al, Nature (1980) 287:193-197 and Scandella and Kornberg, Biochemistry, 10:4447 (1971). Generally with these methods the IFN is not produced in a sufficiently pure form and in sufficiently large quantities for clinical and therapeutic purposes and the resulting IFN preparations produced by recombinant DNA techniques have residual toxic amounts of chemicals, such as sodium dodecyl sulfate (SDS) and other surfactants or preciptants used in the extraction and purification steps which are not acceptable for clinical studies in therapeutic applications.
Both recombinant IFN-.beta. and IL-2 are insoluble in solutions which are at a physiological pH of 7 to 8. Therefore, various processes and additives have been devised to solubilize these proteins.
U.S. Pat. No. 4,463,940 to Hanisch et al. discloses a process for formulating interferon by mixing the interferon and normal serum albumin at pH 12.0 for 5 minutes and then adjusting the pH to 7.5 to obtain a soluble mixture. Exposing the interferon to such a high pH may adversely affect the properties of the interferon. In addition, the formulation contains some SDS.
The only alternatives to the high pH albumin formulation are the addition of detergents or of chaotropic agents such as urea or guanidine hydrochloride to the formulation to solubilize the protein. A detergent such as sodium dodecyl sulfate will cause hemolysis upon injection if its concentration is sufficiently high. Additionally, even a relatively low level of SDS where approximately one molecule of SDS is bound to each molecule of IL-2 may cause liver damage if the IL-2 formulation is administered at very high doses. Chaotropic agents such as urea work to solubilize IL-2, but must be used at very high concentrations of 30% or higher solutions. In addition, with urea there are problems with chemical modification of the protein at high concentrations.
A reagent-grade IL-2 which is in solution with the detergent N-lauryl sarcosinate has been commercialized. In addition, an IL-2 formulation is being sold which contains reagent-grade IL-2 and is recommended to be reconstituted in a complete tissue culture medium with 0.5% deoxycholate, another detergent. Such formulations, however, are not suitable for parenteral injection into humans.
Fatty acids have been used as pharmaceutical absorption promoters for oral and vaginal administration and for rectal application. See, e.g., Jap. Kokai Nos. 8288126 and 8280314 to Kyoto Pharmaceutical Industries. See also Canadian Pat. No. 1113926 and Jap. Kokai 81104812 to Sankyo Co., Ltd. In addition, fatty acids are known for use in topical creams (U.S. Pat. No. 4,331,653), in effecting dissolution of solutes in water (U.S. Pat. No. 3,667,929) and for pharmaceutical injection (U.S. Pat. No. 3,658,970-lauric acid).
Wang et al, J. Parenteral Drug Assoc, 34, 452-462 (1980) provides a review of excipients and pH's for parenteral products used in the United States. This article indicates that it is not trivial to choose excipients for parenteral products as for other dosage forms due to concerns for safety and feasibility in sterilization. Acceptance of a substance to be used as excipient often involves lengthy safety testing or production trials. Also, for reasons of stability or solubility the pH of the product cannot always be adjusted to physiological pH of 7.4. A list of solubilizing agents such as detergents and lipids in use for various drugs is provided on p. 454 and a list of stablizers, including sodium caprylate (octanoate) for normal serum albumin, is provided on p. 458.
There is a need in the art for a protein formulation where the protein is not subjected to high alkalinity so as to alter the protein, the protein is soluble and the formulation is free or virtually free of SDS.